Mechanisms of antimicrobial susceptibility

ABSTRACT

Disclosed herein are methods and compositions for determining the presence or absence of a mechanism of antimicrobial resistance in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/161,857, filed on May 23, 2016, which claims the benefit of priorityof U.S. Provisional Patent Application No. 62/166,964, filed May 27,2015, and is related to U.S. patent application Ser. No. 14/550,335,filed Nov. 21, 2014; the entire disclosures of all are herebyincorporated by reference, in their entireties, for all purposes.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods and compositions for determiningmechanisms that impart an antimicrobial susceptibility phenotype of anorganism.

Description of the Related Art

The antimicrobial susceptibility phenotype of an organism may be due toa variety of mechanisms including, for example, the expression ofvarious enzymes that deactivate the antimicrobial of interest, or cellsurface modifications that prevent entry of the antimicrobial.

Determination of a specific mechanism involved in an antimicrobialsusceptibility phenotype can be important for epidemiological analysisand in determining an appropriate therapy. For example, if it isdetermined that a bacteria is non-susceptible to a β-lactam due to theexpression of a β-lactamase enzyme, this information can be used toinform the possibility of administering a therapy that includes aβ-lactamase inhibitor. If instead the organism is non-susceptible to theβ-lactam and it is determined that the organism does not express theβ-lactamase, this information can be used to determine that aβ-lactamase inhibitor may not provide a suitable therapy.

Different technologies in the art may be used for determining thespecific mechanisms involved in an antimicrobial susceptibilityphenotype. For example, nucleic acid amplification techniques (NAA) suchas polymerase chain reaction (PCR) can be used for detecting thepresence of a gene such as a species-specific gene and a β-lactamasegene. However, such NAA techniques can only infer the possibility anorganism expressing the gene. As such, NAA techniques can identify thepresence of an identifying nucleic acid sequence or antimicrobialresistance gene of an organism but cannot determine if that gene isexpressed and cannot determine the susceptibility of the organism to anantimicrobial.

Colorimetric assays have been developed for the detection of theexpression of enzymes that inactivate an antimicrobial agent. The CarbaNP test is one such example through which the presence of expressedcarbapenemases can be detected due to a color change of a solution(Nordmann, P., L. Poirel, and L. Dortet, Rapid Detection ofCarbapenemase-producing Enterobacteriaceae. Emerging InfectiousDiseases, 2012. 18(9): p. 1503-1507). This test detects the presence ofexpressed carbapenemases and correlates to the carbapenem susceptibilityof an organism. However, the assay cannot determine the identity of theorganism and thus requires prior isolation of the organism and cannotdetermine susceptibility if the mechanism responsible is not enzymatic.

Automated systems exist that provide identification and susceptibilityinformation in an integrated system. The combination of peptide nucleicacid (PNA) fluorescence in situ hybridization (FISH) for organismidentification and automated microscopy for the monitoring of theorganism's growth rate in the presence of antibiotics is one example ofsuch a system (Metzger, S., R. A. Frobel, and W. M. Dunne Jr, Rapidsimultaneous identification and quantitation of Staphylococcus aureusand Pseudomonas aeruginosa directly from bronchoalveolar lavagespecimens using automated microscopy. Diagnostic Microbiology andInfectious Disease, 2014. 79(2): p. 160-165). However, this system lacksthe ability to determine the mechanisms involved in the observedsusceptibility phenotype.

Finally, traditional culture techniques may be used to identifyorganisms and determine the mechanisms involved in imparting asusceptibility phenotype. An example of such an assay is the ModifiedHodge Test used for determining the presence of carbapenemases (Lee, K.,et al., Modified Hodge and EDTA-disk synergy tests to screenmetallo-β-lactamase-producing strains of Pseudomonas and Acinetobactetspecies. Clinical Microbiology and Infection, 2001. 7(2): p. 88-91).However, these techniques rely on observations derived from theantibiotic susceptibility phenotype of an organism and cannot detect thepresence of a mechanism involved in the phenotype if the phenotype isnot expressed.

The limitations of existing technologies thus requires information frommultiple assays and systems in order to provide identification,phenotypic susceptibility determination, and determination of themechanisms involved in imparting the susceptibility phenotype. Becauseof this, healthcare providers must purchase and operate differentsystems in order to acquire this information—a costly and complicatedendeavor. Due to these limitations, there is a need for a single assaythat provides identification, phenotypic susceptibility determination,and determination of the mechanisms involved in imparting thesusceptibility phenotype.

SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions for determining thepresence or absence of a mechanism of antimicrobial resistance in asample. In one embodiment, the present invention relates to a method fordetermining the presence or absence of a mechanism of antimicrobialresistance in a microorganism that is not susceptible to anantimicrobial in a sample, comprising, providing a sample comprising amicroorganism of interest; contacting a portion of said sample with aninhibitor, wherein said inhibitor inhibits a mechanism of conferringnon-susceptibility in said microorganism; contacting said portion ofsaid sample comprising said inhibitor with an antimicrobial agent,wherein said antimicrobial agent is a compound that kills, inhibits thegrowth, or otherwise compromises the viability of one or moremicroorganisms; contacting said portion of said sample with a live cellreporter capable of generating a detectable signal; and detecting thepresence or absence of a signal from said portion of said sample,wherein the presence of said signal indicates that the mechanism ofantimicrobial resistance inhibited by said inhibitor is not present insaid microorganism, and wherein the absence of said signal indicatesthat the mechanism of antimicrobial resistance inhibited by saidinhibitor is present in said microorganism.

In another embodiment, the present invention relates to a method fordetermining the presence or absence of a mechanism of antimicrobialresistance in a microorganism in a sample, comprising, providing asample comprising a microorganism of interest; contacting a firstportion of said sample with an antimicrobial agent, wherein saidantimicrobial agent is a compound that kills, inhibits the growth, orotherwise compromises the viability of one or more microorganisms;contacting said first portion of said sample with a live cell reportercapable of generating a detectable signal; detecting the presence orabsence of a signal from said first portion of said sample comprisingsaid antimicrobial agent and said live cell reporter, wherein theabsence of said signal indicates that said microorganism is susceptibleto said antimicrobial agent, and wherein the presence of said signalindicates that said microorganism is non-susceptible to saidantimicrobial agent; contacting a second portion of said sample with aninhibitor, wherein said inhibitor inhibits a mechanism of conferringnon-susceptibility to said antimicrobial agent in said microorganism;contacting said second portion of said sample comprising said inhibitorwith said antimicrobial agent; contacting said second portion of saidsample with a live cell reporter capable of generating a detectablesignal; and detecting the presence or absence of a signal from saidsecond portion of said sample, wherein the presence of said signalindicates that the mechanism of antimicrobial resistance inhibited bysaid inhibitor is not present in said microorganism, and wherein theabsence of said signal indicates that the mechanism of antimicrobialresistance inhibited by said inhibitor is present in said microorganism.

In still another embodiment, the present invention relates to a methodfor determining the presence or absence of a mechanism of antimicrobialresistance in a microorganism in a sample, comprising, providing asample comprising a microorganism of interest; contacting a firstportion of said sample with an antimicrobial agent and a live cellreporter capable of generating a detectable signal when in the presenceof a substrate, wherein said antimicrobial agent is a compound thatkills, inhibits the growth of, or otherwise compromises the viability ofone or more microorganisms; contacting a second portion of said samplewith a caged substrate and a live cell reporter capable of generating adetectable signal, wherein the caged substrate is un-caged by amechanism associated with a non-susceptibility phenotype to saidantimicrobial agent; and detecting the presence or absence of a signalfrom said first portion and from said second portion, wherein theabsence of said signal from said first portion indicates that themicroorganism is susceptible to said antimicrobial agent, wherein thepresence of said signal from said first portion indicates that themicroorganism is non-susceptible to said antimicrobial agent, whereinthe presence of said signal from said second portion indicates thepresence of said mechanism associated with said non-susceptibilityphenotype to said antimicrobial agent, and wherein the absence of saidsignal from said second portion indicates the absence of said mechanismassociated with said non-susceptibility phenotype to said antimicrobialagent.

In another embodiment, the present invention relates to a kit for fordetermining a mechanism of antimicrobial resistance for a microorganismto an antimicrobial agent comprising, an antimicrobial agent, whereinthe antimicrobial agent is a compound that kills, inhibits the growthof, or otherwise compromises the viability of the growth of one or moremicroorganisms; a non-replicative transduction particle (NRTP)comprising a reporter nucleic acid molecule encoding a reportermolecule; a caged substrate capable of entering the microorganism andbecoming an un-caged substrate in the presence of an enzyme related to amechanism of resistance to the antimicrobial agent, wherein the un-cagedsubstrate reacts with the reporter molecule to produce a detectablesignal, wherein detection of the detectable signal confirms a presenceof the microorganism in the sample; and instructions for using theantimicrobial agent, the NRTP, and the caged substrate to determine themechanism of antimicrobial resistance for the microorganism to theantimicrobial agent based on the presence or absence of a detectableindication of viability associated with the microorganism when themicroorganism is in contact with the antimicrobial agent, the NRTP andthe caged substrate, wherein the presence of a detectable indication ofviability indicates that the microorganism is viable and that the enzymerelated to the mechanism of resistance to the antimicrobial agent isexpressed by the microorganism, and wherein the absence of an indicationof viability indicates that the microorganism is not viable and that theenzyme related to the mechanism of resistance to the antimicrobial agentis not expressed by the microorganism.

In yet another embodiment, the present invention relates to acomposition comprising a meropenem-caged decanol.

In another embodiment, the present invention relates to an in vitro cellculture, a plurality of cells in the cell culture comprising anon-replicative transduction particle (NRTP) comprising a reporternucleic acid molecule encoding a reporter molecule; and a cagedsubstrate capable of becoming uncaged in the presence of enzymaticactivity associated with a mechanism of antimicrobial resistance,wherein said uncaged substrate is capable of reacting with said reportermolecule to produce a detectable signal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 illustrates a reporter system for the detection of intracellularenzymes within viable cells that employs caged substrate molecules thatcan be un-caged by a target intracellular enzyme, according to anembodiment.

FIG. 2 shows % RLU signal from NRTP detection of NDM-1-expressing E.coli in the presence of various additives, normalized to signal from NoAntibiotic.

FIG. 3 shows % RLU signal from NRTP detection of KPC-expressing E. coliin the presence of various additives, normalized to signal from NoAntibiotic

FIG. 4 illustrates a bacterial luciferase-mediated luminescencereaction, according to an embodiment.

FIG. 5 illustrates the synthesis of a meropenem-caged decanal, accordingto an embodiment.

FIG. 6 illustrates an alternative mechanism of synthesis of ameropenem-caged decanal, according to another embodiment.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein, “reporter nucleic acid molecule” refers to a nucleotidesequence comprising a DNA or RNA molecule. The reporter nucleic acidmolecule can be naturally occurring or an artificial or syntheticmolecule. In some embodiments, the reporter nucleic acid molecule isexogenous to a host cell and can be introduced into a host cell as partof an exogenous nucleic acid molecule, such as a plasmid or vector. Incertain embodiments, the reporter nucleic acid molecule can becomplementary to a target gene in a cell. In other embodiments, thereporter nucleic acid molecule comprises a reporter gene encoding areporter molecule (e.g., reporter enzyme, protein). In some embodiments,the reporter nucleic acid molecule is referred to as a “reporterconstruct” or “nucleic acid reporter construct.”

A “reporter molecule” or “reporter” refers to a molecule (e.g., nucleicacid or protein) that confers onto an organism a detectable orselectable phenotype. The detectable phenotype can be colorimetric,fluorescent or luminescent, for example. Reporter molecules can beexpressed from reporter genes encoding enzymes mediating luminescencereactions (luxA, luxB, luxAB, luc, ruc, nluc), genes encoding enzymesmediating colorimetric reactions (lacZ, HRP), genes encoding fluorescentproteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infraredfluorescent proteins), nucleic acid molecules encoding affinity peptides(His-tag, 3X-FLAG), and genes encoding selectable markers (ampC, tet(M),CAT, erm). The reporter molecule can be used as a marker for successfuluptake of a nucleic acid molecule or exogenous sequence (plasmid) into acell. The reporter molecule can also be used to indicate the presence ofa target gene, target nucleic acid molecule, target intracellularmolecule, or a cell, as described herein. Alternatively, the reportermolecule can be a nucleic acid, such as an aptamer or ribozyme.

In some aspects of the invention, the reporter nucleic acid molecule isoperatively linked to a promoter. In other aspects of the invention, thepromoter can be chosen or designed to contribute to the reactivity andcross-reactivity of the reporter system based on the activity of thepromoter in specific cells (e.g., specific species) and not in others.In certain aspects, the reporter nucleic acid molecule comprises anorigin of replication. In other aspects, the choice of origin ofreplication can similarly contribute to reactivity and cross-reactivityof the reporter system, when replication of the reporter nucleic acidmolecule within the target cell contributes to or is required forreporter signal production based on the activity of the origin ofreplication in specific cells (e.g., specific species) and not inothers. In some embodiments, the reporter nucleic acid molecule forms areplicon capable of being packaged as concatameric DNA into a progenyvirus during virus replication.

As used herein, a “target transcript” refers to a portion of anucleotide sequence of a DNA sequence or an mRNA molecule that isnaturally formed by a target cell including that formed during thetranscription of a target gene and mRNA that is a product of RNAprocessing of a primary transcription product. The target transcript canalso be referred to as a cellular transcript or naturally occurringtranscript.

As used herein, the term “transcript” refers to a length of nucleotidesequence (DNA or RNA) transcribed from a DNA or RNA template sequence orgene. The transcript can be a cDNA sequence transcribed from an RNAtemplate or an mRNA sequence transcribed from a DNA template. Thetranscript can be protein coding or non-coding. The transcript can alsobe transcribed from an engineered nucleic acid construct.

A transcript derived from a reporter nucleic acid molecule can bereferred to as a “reporter transcript.” The reporter transcript caninclude a reporter sequence and a cis-repressing sequence. The reportertranscript can have sequences that form regions of complementarity, suchthat the transcript includes two regions that form a duplex (e.g., anintermolecular duplex region). One region can be referred to as a“cis-repressing sequence” and has complementarity to a portion or all ofa target transcript and/or a reporter sequence. A second region of thetranscript is called a “reporter sequence” and can have complementarityto the cis-repressing sequence. Complementarity can be fullcomplementarity or substantial complementarity. The presence and/orbinding of the cis-repressing sequence with the reporter sequence canform a conformation in the reporter transcript, which can block furtherexpression of the reporter molecule. The reporter transcript can formsecondary structures, such as a hairpin structure, such that regionswithin the reporter transcript that are complementary to each other canhybridize to each other.

“Introducing into a cell,” when referring to a nucleic acid molecule orexogenous sequence (e.g., plasmid, vector, construct), meansfacilitating uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of nucleic acidconstructs or transcripts can occur through unaided diffusive or activecellular processes, or by auxiliary agents or devices including via theuse of bacteriophage, virus, and transduction particles. The meaning ofthis term is not limited to cells in vitro; a nucleic acid molecule mayalso be “introduced into a cell,” wherein the cell is part of a livingorganism. In such instance, introduction into the cell will include thedelivery to the organism. For example, for in vivo delivery, nucleicacid molecules, constructs or vectors of the invention can be injectedinto a tissue site or administered systemically. In vitro introductioninto a cell includes methods known in the art, such as electroporationand lipofection. Further approaches are described herein or known in theart.

An “antimicrobial susceptibility phenotype” refers to mechanisms thatare involved in imparting susceptibility or resistance of an organism toan antimicrobial.

An “antimicrobial agent” refers to a compound that kills or inhibits thegrowth or otherwise compromises the viability of one or moremicroorganisms. Antimicrobial agents include antibiotics, antifungals,antiprotozoals, antivirals, and other compounds. “Antimicrobialsusceptibility” or “antimicrobial sensitivity” is the susceptibility ofmicroorganisms to antimicrobial agents. “Non-susceptibility” or“antimicrobial non-susceptibility” arises when a microorganism becomesmore or fully resistant to antimicrobials which previously could treatit. In certain aspects, antimicrobial non-susceptibility is anantibiotic resistance, which applies to bacteria and antibiotics.Antimicrobial non-susceptibility may arise through different ways:natural resistance in certain types of microorganism, genetic mutation,or by one species acquiring resistance from another. Herein,antimicrobial non-susceptibility can appear spontaneously due to randommutations, or more commonly, following gradual buildup over time, andbecause of misuse of antibiotics or antimicrobials. Further, all classesor microorganisms may develop antimicrobial non-susceptibility (e.g.fungi and antifungal resistance; viruses and antiviral resistance;protozoa and antiprotozoal resistance; bacteria and antibioticresistance).

A “transduction particle” refers to a virus capable of delivering anon-viral nucleic acid molecule into a cell. The virus can be abacteriophage, adenovirus, etc.

A “non-replicative transduction particle” (NRTP) refers to a viruscapable of delivering a non-viral nucleic acid molecule into a cell, butdoes not package its own replicated viral genome into the transductionparticle. The virus can be a bacteriophage, adenovirus, etc. NRTPs andmethods of making the same are described in detail in PCT/US2014/026536,filed on Mar. 13, 2014, which is incorporated by reference in itsentirety.

A “plasmid” is a small DNA molecule that is physically separate from,and can replicate independently of, chromosomal DNA within a cell. Mostcommonly found as small circular, double-stranded DNA molecules inbacteria, plasmids are sometimes present in archaea and eukaryoticorganisms. Plasmids are considered replicons, capable of replicatingautonomously within a suitable host.

A “vector” is a nucleic acid molecule used as a vehicle to artificiallycarry foreign genetic material into another cell, where it can bereplicated and/or expressed.

A “virus” is a small infectious agent that replicates only inside theliving cells of other organisms. Virus particles (known as virions)include two or three parts: i) the genetic material made from either DNAor RNA molecules that carry genetic information; ii) a protein coat thatprotects these genes; and in some cases, iii) an envelope of lipids thatsurrounds the protein coat. When referring to a virus that infectsbacteria, the terms “virus”, “phage” and “bacteriophage” are usedinterchangeably in the specification.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., a disease state, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, the term “complementary,” when used to describe a firstnucleotide sequence in relation to a second nucleotide sequence, refersto the ability of an oligonucleotide or polynucleotide comprising thefirst nucleotide sequence to hybridize and form a duplex structure undercertain conditions with an oligonucleotide or polynucleotide comprisingthe second nucleotide sequence, as will be understood by the skilledperson. Complementary sequences are also described as binding to eachother and characterized by binding affinities.

For example, a first nucleotide sequence can be described ascomplementary to a second nucleotide sequence when the two sequenceshybridize (e.g., anneal) under stringent hybridization conditions.Hybridization conditions include temperature, ionic strength, pH, andorganic solvent concentration for the annealing and/or washing steps.The term stringent hybridization conditions refers to conditions underwhich a first nucleotide sequence will hybridize preferentially to itstarget sequence, e.g., a second nucleotide sequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationconditions are sequence dependent, and are different under differentenvironmental parameters. Generally, stringent hybridization conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the nucleotide sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the first nucleotide sequences hybridize to a perfectlymatched target sequence. An extensive guide to the hybridization ofnucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, chap. 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).Other conditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween two strands of a dsRNA, or between the antisense strand of adsRNA and a target sequence, between complementary strands of a singlestranded RNA sequence or a single stranded DNA sequence, as will beunderstood from the context of their use.

As used herein, a “duplex structure” comprises two anti-parallel andsubstantially complementary nucleic acid sequences. Complementarysequences in a nucleic acid construct, between two transcripts, betweentwo regions within a transcript, or between a transcript and a targetsequence can form a “duplex structure.” In general, the majority ofnucleotides of each strand are ribonucleotides, but as described indetail herein, each or both strands can also include at least onenon-ribonucleotide, e.g., a deoxyribonucleotide and/or a modifiednucleotide. The two strands forming the duplex structure may bedifferent portions of one larger RNA molecule, or they may be separateRNA molecules. Where the two strands are part of one larger molecule,and therefore are connected by an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting RNA chain isreferred to as a “hairpin loop.” Where the two strands are connectedcovalently by means other than an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting structure isreferred to as a “linker.” The RNA strands may have the same or adifferent number of nucleotides. The maximum number of base pairs is thenumber of nucleotides in the shortest strand of the duplex minus anyoverhangs that are present in the duplex. Generally, the duplexstructure is between 15 and 30 or between 25 and 30, or between 18 and25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 basepairs in length. In one embodiment the duplex is 19 base pairs inlength. In another embodiment the duplex is 21 base pairs in length.When two different siRNAs are used in combination, the duplex lengthscan be identical or can differ.

As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example a target sequence, as defined herein. Where theregion of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to produce a detectablesignal from a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

II. NRTPs and Reporter Assays

Non-replicative transduction particles (NRTPs) and methods of producingNRTPs are described in PCT Application No. PCT/US2014/026536, filed onMar. 13, 2014 and in U.S. patent application Ser. No. 14/550,335, filedon Nov. 21, 2014, the entire disclosures of both are incorporated byreference in their entireties for all purposes. In some embodiments,NRTPs are produced in a bacterial cell packaging system usingDisruption/Complementation-based methods. This non-replicativetransduction particle packaging system is based on introducing either asilent mutation or a deletion into a component of the genome of avirus/bacteriophage that is recognized by the viral/phage packagingmachinery as the element from which genomic packaging is initiatedduring viral/phage production. Examples of such an element include thepac-site sequence of pac-type bacteriophages and the cos-site sequenceof cos-type bacteriophages.

Because these packaging initiation sites are often found within codingregions of genes that are essential to virus/bacteriophage production,the silent mutation or the deletion is introduced such that the pac-siteis no longer recognized as a site of packaging initiation by theviral/phage packaging machinery. At the same time, the mutation ordeletion does not disrupt the gene in which the site is encoded. Byrendering the packaging site sequence non-functional, the mutatedvirus/bacteriophage is able to undergo a lytic cycle, but is unable topackage its genomic DNA into its packaging unit.

An exogenous reporter nucleic acid molecule, such as plasmid DNA, can beintroduced into a host bacteria cell that has been lysogenized with aviral/phage genome with a non-functional packaging initiation sitesequence. The exogenous reporter nucleic acid molecule can include anative functional packaging initiation site sequence. The exogenousreporter nucleic acid molecule can be introduced into the host bacteriacell and replicated in the cell. When the mutated virus/bacteriophage isundergoing a lytic cycle, the expressed viral/phage packaging machinerypackages the exogenous reporter nucleic acid molecule with thefunctional packaging initiation site sequence into the viral packagingunit. The viral/phage genome is not packaged into the packaging unitbecause its packaging initiation site sequence has been mutated.

Therefore, the present invention contemplates the use of a bacterialcell packaging system for packaging a reporter nucleic acid moleculeinto a NRTP for introduction into a cell, which comprises a hostbacteria cell, a first nucleic acid construct inside the host bacteriacell, comprising of a bacteriophage genome having a non-functionalpackaging initiation site sequence, wherein the non-functional packaginginitiation site sequence prevents packaging of the bacteriophage genomeinto the NRTP, and a second nucleic acid construct inside the hostbacteria cell and separate from the first nucleic acid construct,comprising of the reporter nucleic acid molecule having a reporter geneand a functional packaging initiation site sequence for facilitatingpackaging of a replicon of the reporter nucleic acid molecule into theNRTP, wherein the functional second packaging initiation site sequenceon the second nucleic acid construct complements the non-functionalpackaging initiation site sequence in the bacteriophage genome on thefirst nucleic acid construct. This results in a NRTP that contains thereporter nucleic acid molecule encoding the reporter gene but lacks thebacteriophage genome.

In some embodiments, constructs of the invention (including NRTPs)comprise a reporter nucleic acid molecule including a reporter gene. Thereporter gene can encode a reporter molecule, and the reporter moleculecan be a detectable or selectable marker. In certain embodiments, thereporter gene encodes a reporter molecule that produces a detectablesignal when expressed in a cell.

In certain embodiments, the reporter molecule can be a fluorescentreporter molecule, such as, but not limited to, a green fluorescentprotein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), blue fluorescent protein (BFP), redfluorescent protein (RFP) or mCherry, as well as near-infraredfluorescent proteins.

In other embodiments, the reporter molecule can be an enzyme mediatingluminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc, etc.).Reporter molecules can include a bacterial luciferase, a eukaryoticluciferase, an enzyme suitable for colorimetric detection (lacZ, HRP), aprotein suitable for immunodetection, such as affinity peptides(His-tag, 3X-FLAG), a nucleic acid that function as an aptamer or thatexhibits enzymatic activity (e.g., aptazyme, DNAzyme, ribozyme), or aselectable marker, such as an antibiotic resistance gene (ampC, tet(M),CAT, erm). Other reporter molecules known in the art can be used forproducing signals to detect target nucleic acids or cells.

Disclosed herein are systems for the detection of intracellular enzymeswithin viable cells that employs caged substrate molecules that can beun-caged by a target intracellular enzyme, according to an embodiment ofthe invention.

FIG. 1 depicts the general design and function of an intracellularenzyme detection system. A reporter molecule-expressing vector 101 isdelivered to a target cell 102 with a NRTP (not shown). The reportermolecule-expressing vector 101 is able to penetrate the target cell 102via the NRTP and deliver a reporter molecule gene 103 into the targetcell 102, and a reporter molecule 104 can then be expressed from thereporter molecule gene 103. A caged substrate 105 is also added to thetarget cell 102 and is able to penetrate into the target cell 102. If atarget intracellular enzyme 107 is present in the target cell 106, theenzyme 107 is able to remove the caging component of the caged substrate105, thus producing an un-caged substrate 108. The un-caged substrate108 can then react with the reporter molecule 104 inside of the cell102, and the product of this reaction results in a detectable signal109.

Target cells and enzymes: Target cells can include eukaryotic andprokaryotic cell targets and associated enzymes, including, for example,carbapenemase in Enterobacteriaceae, or β-lactamases in S. aureus.

Vector delivery systems: The delivery of the vector containing therecombinant DNA can by performed by abiologic or biologic systems.Including but not limited to liposomes, virus-like particles,transduction particles derived from phage or viruses, and conjugation.

Reporter molecules and caged substrates: Various reporter molecules andcaged substrates can be employed as those described in Daniel Sobek, J.R., Enzyme detection system with caged substrates, 2007, Zymera, Inc.

III. Antimicrobial Susceptibility Phenotype Determination

Disclosed herein is an integrated system that provides identification,phenotypic susceptibility determination, and determination of themechanisms involved in imparting the susceptibility phenotype to anorganism. The system is based on a live cell reporter system combinedwith reagents that inhibit the activity of mechanisms involved inimparting the susceptibility phenotype as well as reagents whoseactivities are dependent on the mechanisms involved in imparting thesusceptibility phenotype.

In an embodiment, a live cell reporter assay specific to a target cellis used to report on the presence of a target organism. The reporterassay employs a reagent for the production of a detectable signal, suchthat detection of the signal indicates that the target organism ispresent. The reporter assay can also be run combined with anantimicrobial agent to determine if the organism is non-susceptible tothe antimicrobial agent. Additionally, the reporter assay can be runwith a variant of the reagent whose activity is dependent on themechanism involved in imparting the susceptibility phenotype. In anembodiment, the reagent is an inhibitor of the mechanism involved inimparting the susceptibility phenotype. The reporter assay is also runwith the antimicrobial agent in combination with a second reagent thatinhibits a mechanism involved in imparting the susceptibility phenotype.

An example of results produced using the live cell reporter assay arelisted in Table 1 below, where “x” indicates that the reporter assayproduced a positive result. Based on these results, the method indicatesthat Sample 1 contains the target organism that is non-susceptible tothe antimicrobial agent. Sample 2 contains the target organism, it isnon-susceptible to the antimicrobial agent, and the inhibitor inhibitsthe mechanism involved in imparting the susceptibility phenotype. Sample3 contains the target organism that is non-susceptible to theantimicrobial agent and the organism expresses a mechanism that may beresponsible for the antimicrobial phenotype. Sample 4 contains thetarget organism that is non-susceptible to the antimicrobial agent andthe organism does not express the mechanism that may be responsible forthe antimicrobial phenotype. Sample 5 contains the target organism thatis susceptible to the antimicrobial agent. Sample 6 contains the targetorganism, it is susceptible to the antimicrobial agent and it expressesa mechanism that may be involved in an antimicrobial phenotype; as such,the system may determine if an organism expresses a mechanism involvedin a non-susceptible phenotype when an organism exhibits a susceptiblephenotype. Finally, Sample 7 does not contain the target organism

TABLE 1 Exemplary results produced from live cell reporter assayReporter + Reporter + Antimicrobial + Reporter + Reporter AntimicrobialInhibitor Reagent Sample 1 x x x NA Sample 2 x x NA Sample 3 x x NA xSample 4 x x NA Sample 5 x NA Sample 6 x NA x Sample 7 NA

The reporter assay can be employed with a plurality of different livecell reporter systems, a plurality of different antimicrobial agents,and a plurality of different mechanism-conditional reagents,individually or in combination. By evaluating samples with a series ofcombinations of the components of the method, information about thetypes of organisms present in a sample, their antimicrobialsusceptibility to various antimicrobials, and the various resistancemechanisms that may be involved in the susceptibility phenotypes can bededuced.

In some embodiments, the method does not require isolation of theorganism for the assay to be effective. In other embodiments, the methoddoes not require a culture of the organism. In an embodiment, the methodis employed directly from a clinical, environmental, or industrialsample. In an embodiment, assay results are determined within eighthours of running the assay.

Various live cell reporter genes and proteins are known in the art. Forexample, live cell reporter genes and proteins based on thedetermination of absorbance, fluorescence or bioluminescence have beendeveloped to monitor cell viability and proliferation. In some aspects,a reporter gene is fused to one or more regulatory elements (e.g.promotors or enhancers) and the amount of the reporter protein expressedmay be measured. The selection of the reporter system depends on thetarget cell to be analyzed, sensitivity needs and the availabledetection strategy, be it absorbance, fluorescence or bioluminescence.Proteins that traditionally have been used as live cell reportersinclude β-galactosidase (lacZ), chloramphenyl acetyltransferase (CAT),β-glucuronidase (GUS), fluorescent proteins (green, yellow or redfluorescent protein [GFP, YFP or RFP], respectively) and secretoryalkaline phosphatase (SEAP). Further, a variety of luciferases may beused as live cell reporter proteins because of their ultrasensitivedetection capacity and wide dynamic range. In such a live cell reportergene construct, a genetic regulatory element is positioned upstream of aluciferase gene and then the resulting reporter construct is transferredinto animal cells, plant cells, bacteria or other microorganisms, e.g.,through transfection, transformation, transduction or injection.Expression of the luciferase reporter gene is then measured to quantifythe activity of the regulatory element and/or to determine cellviability. Luciferase enzymes can be used as single reporters to studyone biological event in a given experiment, but because of theirdifferent spectral properties and/or substrates, multiple luciferaseenzymes can be combined for multiplex luciferase experiments. In certainaspects, the reporter gene may be selected from the group consisting ofgenes encoding enzymes mediating luminescence reactions (luxA, luxB,luxAB, luc, rue, nluc), genes encoding enzymes mediating colorimetricreactions (lacZ, HRP), genes encoding fluorescent proteins (GFP, eGFP,YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins),nucleic acid molecules encoding affinity peptides (His-tag, 3X-FLAG),and genes encoding selectable markers (ampC, tet(M), CAT, erm).

In certain aspects, a reporter nucleic acid molecule encoding a reportermolecule may be packaged into a non-replicative transduction particle(NRTP) for introduction into a target cell, e.g. a microbial orbacterial cell. Herein, a live cell reporter comprises a non-replicativetransduction particle (NRTP) comprising a reporter nucleic acid moleculeencoding a reporter molecule. More specifically, the NRTP comprises areporter gene that is operably linked to an inducible promoter thatcontrols the expression of a target gene within a target cell. Thereporter nucleic acid molecule can thus be introduced into the targetcell via a NRTP. When the NRTP that includes the reporter gene isintroduced into the target cell, expression of the reporter gene ispossible via induction of the target gene promoter in the reporternucleic acid molecule. In certain aspects, the live cell reportercomprises a nucleic acid molecule comprising a light-emittingprotein-encoding gene. In some aspects, the light-emittingprotein-encoding gene is a luciferase gene. Suitable exemplarynon-replicative transduction particle packaging systems can be based ondisruption of a component of the genome of a virus that is recognized bythe viral packaging machinery as the element from which genomicpackaging is initiated during viral production. In some aspects, thisdisruption disrupts a packaging initiation site from a bacteriophage,and also disrupts a terminase function. Examples of the disruptedelements include the pac-site sequence of pac-type bacteriophages andthe cos-site sequence of cos-type bacteriophages. When the packaginginitiation site sequence within the phage is disrupted, the phage cannotproduce functional terminases. In an example, the pac-site is encodedwithin a pacA gene sequence, and terminase functions require both afunctional PacA and PacB. Herein, plasmid DNA is packaged into a phagecapsid by complementing said disrupted terminases and including arecognizable packaging initiation site on the plasmid DNA. Thebacteriophage can be any bacteriophage, such as an Enterobacteriaceaebacteriophage PI, an S. aureus bacteriophage φ80α or a bacteriophageφ11.

IV. Methods for Determining a Mechanism of Antimicrobial Resistance

In one embodiment a method for determining the presence or absence of amechanism of antimicrobial resistance in a microorganism that is notsusceptible to an antimicrobial in a sample is provided, comprisingproviding a sample comprising a microorganism of interest; contacting atleast a portion of said sample with an inhibitor, wherein said inhibitorinhibits a mechanism of conferring non-susceptibility in saidmicroorganism; contacting said portion of said sample comprising saidinhibitor with an antimicrobial agent, wherein said antimicrobial agentis a compound that kills, inhibits the growth, or otherwise compromisesthe viability of one or more microorganisms; contacting said portion ofsaid sample with a live cell reporter capable of generating a detectablesignal; and detecting the presence or absence of a signal from saidportion of said sample, wherein the presence of said signal indicatesthat the mechanism of antimicrobial resistance inhibited by saidinhibitor is not present in said microorganism, and wherein the absenceof said signal indicates that the mechanism of antimicrobial resistanceinhibited by said inhibitor is present in said microorganism.

In another embodiment a method for determining the presence or absenceof a mechanism of antimicrobial resistance in a microorganism in asample is provided, comprising providing a sample comprising amicroorganism of interest; contacting a first portion of said samplewith an antimicrobial agent, wherein said antimicrobial agent is acompound that kills, inhibits the growth, or otherwise compromises theviability of one or more microorganisms; contacting said first portionof said sample with a live cell reporter capable of generating adetectable signal; detecting the presence or absence of a signal fromsaid first portion of said sample comprising said antimicrobial agentand said live cell reporter, wherein the absence of said signalindicates that said microorganism is susceptible to said antimicrobialagent, and wherein the presence of said signal indicates that saidmicroorganism is non-susceptible to said antimicrobial agent; contactinga second portion of said sample with an inhibitor, wherein saidinhibitor inhibits a mechanism of conferring non-susceptibility to saidantimicrobial agent in said microorganism; contacting said secondportion of said sample comprising said inhibitor with said antimicrobialagent; contacting said second portion of said sample with a live cellreporter capable of generating a detectable signal; and detecting thepresence or absence of a signal from said second portion of said sample,wherein the presence of said signal indicates that the mechanism ofantimicrobial resistance inhibited by said inhibitor is not present insaid microorganism, and wherein the absence of said signal indicatesthat the mechanism of antimicrobial resistance inhibited by saidinhibitor is present in said microorganism. In certain embodiments themethod further comprises contacting a third portion of said sample witha live cell reporter capable of generating a detectable signal when inthe presence of a caged substrate, wherein said caged substrate isun-caged by a mechanism associated with a non-susceptibility phenotypeto said antimicrobial agent; and detecting the presence or absence of asignal from said third portion of said sample, wherein the presence ofsaid signal indicates that the mechanism associated with saidnon-susceptibility phenotype to said antimicrobial agent is present insaid microorganism, and wherein the absence of said signal indicatesthat the mechanism associated with said non-susceptibility phenotype tosaid antimicrobial agent is not present in said microorganism.

In certain embodiments of these methods said inhibitor comprises a metalchelator or a beta lactamase inhibitor. In some embodiments said metalchelator is EDTA. In some embodiments, said beta lactamase inhibitor isboronic acid. In some embodiments said metal chelator is EDTA and saidbeta lactamase inhibitor is boronic acid.

In another embodiment, a method for determining the presence or absenceof a mechanism of antimicrobial resistance in a microorganism in asample is provided, comprising providing a sample comprising amicroorganism of interest; contacting a first portion of said samplewith an antimicrobial agent and a live cell reporter capable ofgenerating a detectable signal when in the presence of a substrate,wherein said antimicrobial agent is a compound that kills, inhibits thegrowth of, or otherwise compromises the viability of one or moremicroorganisms; contacting a second portion of said sample with a cagedsubstrate and a live cell reporter capable of generating a detectablesignal, wherein the caged substrate is un-caged by a mechanismassociated with a non-susceptibility phenotype to said antimicrobialagent; and detecting the presence or absence of a signal from said firstportion and from said second portion, wherein the absence of said signalfrom said first portion indicates that the microorganism is susceptibleto said antimicrobial agent, wherein the presence of said signal fromsaid first portion indicates that the microorganism is non-susceptibleto said antimicrobial agent, wherein the presence of said signal fromsaid second portion indicates the presence of said mechanism associatedwith said non-susceptibility phenotype to said antimicrobial agent, andwherein the absence of said signal from said second portion indicatesthe absence of said mechanism associated with said non-susceptibilityphenotype to said antimicrobial agent.

In certain embodiments, said caged substrate comprises a fatty aldehyde.In some embodiments, said caged substrate comprises a fatty aldehydecaged by an antimicrobial agent-based molecule. In some embodiments,said fatty aldehyde is uncaged upon contacting an enzyme that reactswith said caged molecule in a manner that allows said fatty aldehyde tointeract with said reporter molecule to produce said signal. Inparticular embodiments, said enzyme is a carbapenemase. In someembodiments, said caged substrate comprises a fatty aldehyde caged by ameropenem-based molecule. In certain embodiments, said fatty aldehyde isdecanal. In certain embodiments, said fatty aldehyde is uncaged uponcontacting a carbapenemase, allowing said fatty acid to interact withsaid reporter molecule to produce said signal. In some embodiments, saidcaged substrate is encapsulated in a liposome.

In certain embodiments of the above methods, contacting said sample withsaid live cell reporter comprises introducing into said sample anon-replicative transduction particle comprising a reporter geneencoding a reporter molecule and lacking a bacteriophage genome underconditions such that said non-replicative transduction particle cantransduce said microorganism and wherein said reporter gene can beexpressed in said microorganism; and providing conditions for activationof said reporter molecule. In some embodiments, said reporter gene isselected from the group consisting of genes encoding enzymes mediatingluminescence reactions, genes encoding enzymes mediating colorimetricreactions, genes encoding fluorescent proteins, genes encodingselectable markers, and nucleic acid molecules encoding affinitypeptides. In certain embodiments, said reporter gene is operativelylinked to a constitutive promoter. In some embodiments, said reportergene is selected from the group consisting of genes encoding enzymesmediating luminescence reactions (luxA, luxB, luxAB, luc, ruc, nluc),genes encoding enzymes mediating colorimetric reactions (lacZ, HRP),genes encoding fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP,mCherry, near-infrared fluorescent proteins), nucleic acid moleculesencoding affinity peptides (His-tag, 3X-FLAG), and genes encodingselectable markers (ampC, tet(M), CAT, erm). In certain embodiments,said genes encoding enzymes mediating luminescence reactions comprisegenes encoding one or more luciferases. In certain embodiments, saidlive cell reporter comprises a nucleic acid molecule comprising alight-emitting protein-encoding gene. In certain embodiments, saidlight-emitting protein-encoding gene is a luciferase gene.

In some embodiments, said reporter signal can be detected from a sampleat a limit of detection (LoD) of less than 1,000 colony forming units(CFU). In some embodiments, said reporter signal can be detected from asample at a limit of detection (LoD) of less than 100 colony formingunits (CFU). In some embodiments, said reporter signal can be detectedfrom a sample at a limit of detection (LoD) of less than 10 colonyforming units (CFU). In some embodiments, said reporter signal can bedetected from a sample at a LoD less than five CFU. In some embodiments,said reporter signal can be detected from a sample at a LoD of three orless CFU.

In certain embodiments of the above methods said antimicrobial agent isselected from the group consisting of: β-lactams, extended-spectrumβ-lactams, Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, anygeneration of Cephalosporins, Glycopeptides, Lincosamides, Lipopeptide,Macrolides, Monobactams, Nitrofurans, Oxazolidonones, Penicillins,Polypeptides, Quinolones, Fluoroquinolones, Streptogramins,Sulfonamides, Tetracyclines, Rifampicin, mycobacterial antibiotics,Chloramphenicol, and Mupirocin.

In certain embodiments of the above methods said microorganism is aprokaryote or a eukaryote. In some embodiments, said prokaryote areGram-negative bacteria or Gram-positive bacteria. In some embodiments,said microorganism is Staphylococcus spp., Enterobacteriaceae,Enterococcus spp. Streptococcus spp., Acinetobacter spp., Pseudomonasspp., Stenotrophomonas spp., and Mycobacterium spp., or Candida.

In certain embodiments, said mechanism of antimicrobial resistancecomprises beta-lactamase activity or carbapenemase activity. In someembodiments, said carbapenemase activity is from a Class Acarbapenemase, a Class B carbapenemase, a class D carbapenemase, or acombination thereof. In some embodiments, said antimicrobial resistancecomprises resistance to a carbapenem.

In another embodiment, a method for determining the presence or absenceof a mechanism of antimicrobial resistance in a microorganism in asample is provided, comprising providing a sample comprising amicroorganism of interest; contacting said sample with a live cellreporter capable of generating a detectable signal, an antimicrobialagent, and an inhibitor, wherein said inhibitor is capable of inhibitinga mechanism of resistance to said antimicrobial agent; and detecting thepresence or absence of said signal, wherein the absence of said signalindicates the presence of said mechanism of antimicrobial resistance,and wherein the presence of said signal indicates the absence of saidmechanism of antimicrobial resistance.

In another embodiment, a method for determining the presence or absenceof a mechanism of antimicrobial resistance in a microorganism in asample is provided, comprising providing a sample comprising amicroorganism of interest; contacting a first portion of said samplewith a live cell reporter capable of generating a detectable signal;contacting said first portion of said sample with an antimicrobialagent; and detecting the presence or absence of said signal in saidfirst portion, wherein the presence of said signal indicates saidmicroorganism comprises a non-susceptible phenotype, and wherein theabsence of said signal indicates said microorganism comprises asusceptible phenotype.

In certain embodiments the method further comprises contacting saidfirst portion of said sample comprising said live cell reporter, saidantimicrobial agent, and said microorganism comprising a resistantphenotype with an inhibitor capable of inhibiting a mechanism ofresistance to said antimicrobial agent; and detecting the presence orabsence of said signal in said first portion, wherein the absence ofsaid signal indicates the presence of a type of said mechanism ofantimicrobial resistance in said microorganism comprising a resistantphenotype, and wherein the presence of said signal indicates the absenceof said type of said mechanism of antimicrobial resistance in saidmicroorganism.

In certain embodiments, the method further comprises contacting a secondportion of said sample comprising with a live cell reporter and a cagedsubstrate under conditions such that the caged substrate entersmicroorganism, wherein the caged substrate is capable of becominguncaged in the presence of enzymatic activity associated with themechanism of antimicrobial resistance, and wherein said uncagedsubstrate is capable of reacting with said live cell reporter to producesaid detectable signal, wherein said live cell reporter will not producesaid detectable signal in the absence of said uncaged substrate; anddetecting the presence or absence of said signal in said second portion,wherein the presence of said signal indicates the presence of saidmechanism of antimicrobial resistance, and wherein the absence of saidsignal indicates the absence of said mechanism of antimicrobialresistance.

In certain embodiments, the method further comprises contacting a secondportion of said sample with a live cell reporter and a caged substrateunder conditions such that the caged substrate enters microorganism,wherein the caged substrate is capable of becoming uncaged in thepresence of enzymatic activity associated with the mechanism ofantimicrobial resistance, wherein said uncaged substrate is capable ofreacting with said reporter molecule to produce said detectable signal,and wherein said live cell reporter will not produce said detectablesignal in the absence of said uncaged substrate; and detecting thepresence or absence of said signal, wherein the presence of said signalindicates the presence of said mechanism of antimicrobial resistance,and wherein the absence of said signal indicates the absence of saidmechanism of antimicrobial resistance.

In certain embodiments, the method further comprises contacting a secondportion of said sample with a live cell reporter and a caged substrateunder conditions such that the caged substrate enters microorganism,wherein the caged substrate is capable of becoming uncaged in thepresence of enzymatic activity associated with the mechanism ofantimicrobial resistance, wherein said uncaged substrate is capable ofreacting with said reporter molecule to produce said detectable signal,and wherein said live cell reporter will not produce said detectablesignal in the absence of said uncaged substrate; contacting said secondportion with an inhibitor capable of inhibiting a mechanism ofresistance to said antimicrobial agent; and detecting the presence orabsence of said signal, wherein the absence of said signal indicates thepresence of a type of said mechanism of antimicrobial resistance in saidmicroorganism comprising a resistant phenotype, and wherein the presenceof said signal indicates the absence of said type of said mechanism ofantimicrobial resistance in said microorganism.

In certain embodiments of these methods, said antimicrobial agent isselected from the group consisting of: β-lactams, extended-spectrumβ-lactams, Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, anygeneration of Cephalosporins, Glycopeptides, Lincosamides, Lipopeptide,Macrolides, Monobactams, Nitrofurans, Oxazolidonones, Penicillins,Polypeptides, Quinolones, Fluoroquinolones, Streptogramins,Sulfonamides, Tetracyclines, Rifampicin, mycobacterial antibiotics,Chloramphenicol, and Mupirocin. In certain embodiments, said inhibitorcomprises a metal chelator or a beta lactamase inhibitor. In certainembodiments, said metal chelator is EDTA. In certain embodiments, saidbeta lactamase inhibitor is boronic acid. In certain embodiments, saidlive cell reporter comprises a nucleic acid molecule comprising alight-emitting protein-encoding gene. In certain embodiments, saidlight-emitting protein-encoding gene is a luciferase gene. In certainembodiments, said caged substrate comprises a fatty aldehyde. In certainembodiments, said caged substrate comprises a fatty aldehyde caged by ameropenem-based molecule. In certain embodiments, said fatty aldehyde isdecanal. In certain embodiments, said fatty aldehyde is uncaged uponcontacting a carbapenemase, allowing said fatty acid to interact withsaid reporter molecule to produce said signal. In certain embodiments,said caged substrate is encapsulated in a liposome.

In another embodiment, a method for determining the presence or absenceof a mechanism of antimicrobial resistance in a microorganism in asample is provided, comprising providing a sample comprising amicroorganism of interest; contacting the sample with a live cellreporter; contacting the sample with a caged substrate under conditionssuch that the caged substrate enters microorganism, wherein the cagedsubstrate is capable of becoming uncaged in the presence of enzymaticactivity associated with a mechanism of antimicrobial resistance, andwherein said uncaged substrate is capable of reacting with said reportermolecule to produce a detectable signal; and detecting the presence orabsence of said signal, wherein the presence of said signal indicatesthe presence of said enzymatic activity associated with a mechanism ofantimicrobial resistance in said microorganism of interest, and whereinthe absence of said signal indicates the absence of said enzymaticactivity associated with a mechanism of antimicrobial resistance in saidmicroorganism of interest.

In certain embodiments, said live cell reporter comprises anon-replicative transduction particle (NRTP) comprising a reporternucleic acid molecule encoding a reporter molecule. In certainembodiments, the method further comprises contacting said sample with anantimicrobial agent to determine whether said microorganism is resistantto said antimicrobial agent. In certain embodiments, said sample iscontacted with the antimicrobial agent prior to contacting the samplewith the live cell reporter. In certain embodiments, said sample iscontacted with the live cell reporter prior to contacting the samplewith the antimicrobial agent. In certain embodiments, said antimicrobialagent is selected from the group consisting of: β-lactams,extended-spectrum β-lactams, Aminoglycosides, Ansamycins, Carbacephem,Carbapenems, any generation of Cephalosporins, Glycopeptides,Lincosamides, Lipopeptide, Macrolides, Monobactams, Nitrofurans,Oxazolidonones, Penicillins, Polypeptides, Quinolones, Fluoroquinolones,Streptogramins, Sulfonamides, Tetracyclines, Rifampicin, mycobacterialantibiotics, Chloramphenicol, and Mupirocin.

In certain embodiments, said microorganism is a prokaryote or aeukaryote.

In certain embodiments, said microorganism is a Gram-negative bacteria.

In certain embodiments, said microorganism is Staphylococcus spp.,Enterobacteriaceae, Enterococcus spp. Streptococcus spp., Acinetobacterspp., Pseudomonas spp., Stenotrophomonas spp., and Mycobacterium spp.,or Candida.

In certain embodiments, said microorganism of interest is resistant toat least one antimicrobial agent. In certain embodiments, said live cellreporter comprises a nucleic acid molecule comprising a light-emittingprotein-encoding gene. In certain embodiments, said light-emittingprotein-encoding gene is a luciferase gene. In certain embodiments, saidenzymatic activity comprises beta-lactamase activity. In certainembodiments, said enzymatic activity comprises carbapenemase activity.In certain embodiments, said carbapenemase activity is from a Class Acarbapenemase, a Class B carbapenemase, a class D carbapenemase, or acombination thereof.

In certain embodiments, said caged substrate comprises a fatty aldehyde.In certain embodiments, the caged substrate comprises a fatty aldehydecaged by a meropenem-based molecule. In certain embodiments, said fattyaldehyde is decanal. In certain embodiments, said fatty aldehyde isuncaged upon contacting a carbapenemase, allowing said fatty acid tointeract with said reporter molecule to produce said signal. In certainembodiments, the caged substrate is encapsulated in a liposome. Incertain embodiments, said antimicrobial resistance comprises resistanceto a carbapenem. In certain embodiments, said carbapenem comprisesertapenem.

In certain embodiments, the method further comprises contacting thesample with an inhibitor suspected of reducing the activity of saidenzyme; and detecting the presence or absence of said signal, wherein areduction of said signal due to addition of said inhibitor as comparedto the signal in the absence of said inhibitor indicates the presenceand activity of said enzyme associated with a mechanism of antimicrobialresistance in said microorganism of interest. In certain embodiments,said inhibitor comprises a metal chelator or a beta lactamase inhibitor.In certain embodiments, said metal chelator is EDTA. In certainembodiments, said beta lactamase inhibitor is boronic acid.

In another embodiment, a method for determining the presence or absenceof an enzyme correlated with a mechanism of antimicrobial resistance ina microorganism in a sample is provided comprising providing a samplecomprising a microorganism of interest; contacting the sample with acaged substrate, the caged substrate capable of becoming uncaged uponcontact with an active enzyme associated with a mechanism ofantimicrobial resistance, wherein said uncaged substrate is capable ofproducing a signal; contacting the sample with an inhibitor targetingsaid enzyme, wherein said inhibitor reduces the activity of said enzyme;and detecting the presence or absence of said signal, wherein theabsence or reduction of said signal indicates inhibition of the activityof said enzyme associated with a mechanism of antimicrobial resistancein said microorganism of interest.

In another embodiment, a method of detecting the presence or absence ofa microorganism in a sample is provided, comprising contacting thesample with a caged substrate, the caged substrate capable of becominguncaged due to enzymatic activity associated with said microorganism,wherein said uncaged substrate is capable of producing a signal; anddetecting the presence or absence of said signal, wherein the presenceof said signal indicates the presence of said microorganism in saidsample, and wherein the absence of said signal indicates the absence ofsaid microorganism in said sample.

In another embodiment, a method for determining a mechanism ofantimicrobial resistance, comprising providing a sample comprising anEnterobacteriaceae; contacting the sample with carbapenem; contactingthe sample with a non-replicative transduction particle (NRTP)comprising a luciferase gene encoding a luciferase; contacting thesample with a decanal molecule caged by a meropenem-based molecule,under conditions such that the caged decanal enters theEnterobacteriaceae and becomes un-caged in the presence ofcarbapenemase, wherein the un-caged decanal reacts with the luciferaseto produce a detectable signal, wherein detection of the detectablesignal confirms a presence of a carbapenemase-based resistance mechanismin the Enterobacteriaceae in the sample. In certain embodiments, themethod further comprises contacting said sample with boronic acid,wherein the reduction of said detectable signal after contacting saidsample with boronic acid confirms the presence of a Class Acarbapenemase resistance mechanism in the Enterobacteriaceae in thesample. In certain embodiments, the method further comprises contactingsaid sample with EDTA, wherein the reduction of said detectable signalafter contacting said sample with EDTA confirms the presence of a ClassB carbapenemase resistance mechanism in the Enterobacteriaceae in thesample.

V. Compositions, Microorganisms and Kits

In one aspect, a composition is provided comprising a meropenem-cageddecanal.

In another aspect, a microorganism is provided comprising anon-replicative transduction particle (NRTP) comprising a reporternucleic acid molecule encoding a reporter molecule; and a cagedsubstrate capable of becoming uncaged in the presence of enzymaticactivity associated with a mechanism of antimicrobial resistance,wherein said uncaged substrate is capable of reacting with said reportermolecule to produce a detectable signal. In certain aspects, themicroorganism further comprises an antimicrobial agent. In certainaspects, the microorganism further comprises an inhibitor of saidenzymatic activity associated with said mechanism of antimicrobialresistance.

In one embodiment, an in vitro cell culture is provided comprising aplurality of cells in the cell culture comprising a non-replicativetransduction particle (NRTP) comprising a reporter nucleic acid moleculeencoding a reporter molecule; and a caged substrate capable of becominguncaged in the presence of enzymatic activity associated with amechanism of antimicrobial resistance, wherein said uncaged substrate iscapable of reacting with said reporter molecule to produce a detectablesignal. In certain aspects, said plurality of cells further comprises anantimicrobial agent. In certain aspects, said plurality of cells furthercomprises an inhibitor of said enzymatic activity associated with saidmechanism of antimicrobial resistance.

In another embodiment, a kit for determining a mechanism ofantimicrobial resistance for a microorganism to an antimicrobial agentis provided comprising an antimicrobial agent, wherein the antimicrobialagent is a compound that kills, inhibits the growth of, or otherwisecompromises the viability of the growth of one or more microorganisms; anon-replicative transduction particle (NRTP) comprising a reporternucleic acid molecule encoding a reporter molecule; a caged substratecapable of entering the microorganism and becoming an un-caged substratein the presence of an enzyme related to a mechanism of resistance to theantimicrobial agent, wherein the un-caged substrate reacts with thereporter molecule to produce a detectable signal, wherein detection ofthe detectable signal confirms a presence of the microorganism in thesample; and instructions for using the antimicrobial agent, the NRTP,and the caged substrate to determine the mechanism of antimicrobialresistance for the microorganism to the antimicrobial agent based on thepresence or absence of a detectable indication of viability associatedwith the microorganism when the microorganism is in contact with theantimicrobial agent, the NRTP and the caged substrate, wherein thepresence of a detectable indication of viability indicates that themicroorganism is viable and that the enzyme related to the mechanism ofresistance to the antimicrobial agent is expressed by the microorganism,and wherein the absence of an indication of viability indicates that themicroorganism is not viable and that the enzyme related to the mechanismof resistance to the antimicrobial agent is not expressed by themicroorganism.

In certain aspects and embodiments of the microorganism, cell cultureand kit, said caged substrate comprises a fatty aldehyde. In someembodiments, said caged substrate comprises a fatty aldehyde caged by anantimicrobial agent-based molecule. In some embodiments, said fattyaldehyde is uncaged upon contacting an enzyme that reacts with saidcaged molecule in a manner that allows said fatty aldehyde to interactwith said reporter molecule to produce said signal. In particularembodiments, said enzyme is a carbapenemase. In some embodiments, saidcaged substrate comprises a fatty aldehyde caged by a meropenem-basedmolecule. In certain embodiments, said fatty aldehyde is decanal. Incertain embodiments, said fatty aldehyde is uncaged upon contacting acarbapenemase, allowing said fatty acid to interact with said reportermolecule to produce said signal. In some embodiments, said cagedsubstrate is encapsulated in a liposome.

In some embodiments, said reporter nucleic acid molecule is selectedfrom the group consisting of genes encoding enzymes mediatingluminescence reactions, genes encoding enzymes mediating colorimetricreactions, genes encoding fluorescent proteins, genes encodingselectable markers, and nucleic acid molecules encoding affinitypeptides. In certain embodiments, said reporter nucleic acid molecule isoperatively linked to a constitutive promoter. In some embodiments, saidreporter nucleic acid molecule is selected from the group consisting ofgenes encoding enzymes mediating luminescence reactions (luxA, luxB,luxAB, luc, ruc, nluc), genes encoding enzymes mediating colorimetricreactions (lacZ, HRP), genes encoding fluorescent proteins (GFP, eGFP,YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent proteins),nucleic acid molecules encoding affinity peptides (His-tag, 3X-FLAG),and genes encoding selectable markers (ampC, tet(M), CAT, erm). Incertain embodiments, said genes encoding enzymes mediating luminescencereactions comprise genes encoding one or more luciferases. In certainembodiments, said live cell reporter comprises a nucleic acid moleculecomprising a light-emitting protein-encoding gene. In certainembodiments, said light-emitting protein-encoding gene is a luciferasegene.

In certain embodiments said antimicrobial agent is selected from thegroup consisting of: β-lactams, extended-spectrum β-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Streptogramins, Sulfonamides,Tetracyclines, Rifampicin, mycobacterial antibiotics, Chloramphenicol,and Mupirocin. In certain embodiments said microorganism is a prokaryoteor a eukaryote. In some embodiments, said prokaryote are Gram-negativebacteria or Gram-positive bacteria. In some embodiments, saidmicroorganism is Staphylococcus spp., Enterobacteriaceae, Enterococcusspp. Streptococcus spp., Acinetobacter spp., Pseudomonas spp.,Stenotrophomonas spp., and Mycobacterium spp., or Candida. In certainembodiments, said mechanism of antimicrobial resistance comprisesbeta-lactamase activity or carbapenemase activity. In some embodiments,said carbapenemase activity is from a Class A carbapenemase, a Class Bcarbapenemase, a class D carbapenemase, or a combination thereof. Insome embodiments, said antimicrobial resistance comprises resistance toa carbapenem.

EXAMPLES Example 1: Detection of NDM-1 via an EDTA inhibitor

Carbapenem non-susceptibility may be imparted by carbapenemase enzymesas well as by other mechanisms including over expression ofnon-carbapenemase β-lactamase enzymes and porin mutations.Carbapenemases are derived from the classes including A, B, and D. ClassA carbapenemases include those encoded by the KPC (K. pneumoniaecarbapenemase) genes and are susceptible to inhibition by β-lactamaseinhibitors. Class B carbapenemases include those encoded by the IMP-type(metallo-β-lactamases), New Delhi metallo-β-lactamase (NDM-1), and VIM(Verona integron-encoded metallo-β-lactamase) genes and require ionicmetal to function. Class D carbapenemases include those encoded by theOXA (oxacillinase) genes and are not susceptible to inhibition byβ-lactamase inhibitors.

An NDM-1 detection assay is produced using a luciferase-expressingnon-replicative transduction particle specific to Enterobacteriaceae (asdescribed in PCT/US2014/026536, Example 1), ertapenem, and the chelatorEthylenediaminetetraacetic acid (EDTA).

A culture of a clinical isolate of an NDM-1-expressing E. coli wasprepared in LB media and used to determine the response of the NRTPreporter in the presence of ertapenem, EDTA, and Zinc. The assay wasprepared by mixing 50 μL of the cell culture at an OD600 of 0.02 to 50μL of LB media and 100 μL of NRTP reagent prepared in a microwell plate.The assay was incubated for 2 hours at 37° C. under the followingconditions: (1) No additives, (2) with the addition of ertapenem at 0.25μg/mL, (3) with the addition of ertapenem and EDTA at 750 μg/mL, and (4)with the addition of ertapenem and Zinc sulfate at 70 μg/mL. After theincubation, the samples were assayed for luminescence after theinjection of a nonanal reagent as a substrate for the live cell reporterusing a Molecular Devices SpectraMax L luminometer.

The luminescence readings were then normalized to those produced bysample (1) and plotted as a percentage relative to sample (1) as shownin FIG. 2.

From the results depicted in FIG. 2, it can be seen that theNDM-1-expressing E. coli was non-susceptible to ertapenem, since theluminescence signal was not significantly decreased in the presence ofthe ertapenem. The signal was significantly decreased in the presence ofEDTA, indicating that the mechanism involved in the ertapenemnon-susceptibility phenotype (i.e. NDM-1) was inhibited in the presenceof the chelating agent. Conversely, upon the addition of Zinc to theassay, the signal was significantly increased, again indicative of therole of NDM-1 in imparting the ertapenem non-susceptibility phenotype.

Example 2: Detection of KPC via a Boronic Acid Inhibitor

In another embodiment of the invention, a KPC detection assay isproduced using a luciferase-expressing non-replicative transductionparticle specific to Enterobacteriaceae as described inPCT/US2014/026536, Example 1, ertapenem and the β-lactamase inhibitorboronic acid.

A culture of a clinical isolate of a KPC-expressing E. coli was preparedin LB media and used to determine the response of the NRTP reporter inthe presence of ertapenem, and boronic acid. The replicates of the assaywere prepared mixing 50 μL of the cell culture at an OD600 of 0.02 to 50μL of LB media and 100 μL of NRTP reagent prepared in a microwell plate.The assay was incubated for 2 hours at 37° C. under the followingconditions: (1) No additives, (2) with the addition of ertapenem at 1μg/mL, and (3) with the addition of ertapenem and phenyl boronic acid400 μg/mL. After the incubation, the samples were assayed forluminescence after the injection of a nonanal reagent as a substrate forthe live cell reporter using a Molecular Devices SpectraMax Lluminometer.

The luminescence readings were then normalized to those produced bysample (1) and plotted as the average percentage relative to sample (1)as shown in FIG. 1.

From the results depicted in FIG. 2, it can be seen that theKPC-expressing E. coli was non-susceptible to ertapenem, since theluminescence signal was not significantly decreased in the presence ofthe ertapenem and in fact the average signal was higher than thatwithout ertapenem. The signal was significantly decreased in thepresence of boronic acid, indicating that the mechanism involved in theertapenem non-susceptibility phenotype (i.e. KPC) was inhibited in thepresence of the β-lactamase inhibitor.

Example 3: Determination of Carbapenem Susceptibility Mechanism

A carbapenem susceptibility detection system (for determiningantimicrobial susceptibility phenotype) is produced based on a bacterialluciferase-expressing non-replicative transduction particle (NRTP)specific to Enterobacteriaceae, and a caged luciferase substrate thatmay be un-caged by a carbapenemase enzyme.

The bacterial luciferase luminescence reaction requires the bacterialluciferase enzyme LuxAB, reduced flavin mononucleotide FMNH2, a fattyaldehyde such as decanal, and molecular oxygen (FIG. 4). In thisreaction, the luciferase enzyme first binds to FMNH2 and mediates areaction between FMNH2 and molecular oxygen. Next, a fatty aldehyde,such as decanal, reacts with this complex and causes oxidation of bothFMNH2 and decanal, resulting in a photon emission.

Fatty aldehydes such as decanal can be caged by linking to them amolecule (such as a meropenem-based molecule). When caged, the fattyaldehyde is unable to mediate a luminescence reaction. When un-caged,the aldehyde is able to mediate a luminescence reaction. A caged fattyaldehyde can be designed to become un-caged in the presence of an enzymethat hydrolyses the caging element.

A reporter system is designed to detect the presence of at least oneintracellular enzyme within viable cells that mediates the un-caging ofthe caged molecule by one or more target intracellular enzyme (asdescribed in PCT/US2014/026536, paragraphs 196-203). In short, areporter molecule-expressing nucleic acid (e.g. a vector) may bedelivered to a target cell with a non-replicative transduction particle(NRTP). Herein, the reporter molecule-expressing nucleic acid is able topenetrate the target cell via the NRTP and deliver a reporter moleculegene into the target cell and a reporter molecule can then be expressedfrom the reporter molecule gene. A caged substrate is also added to thetarget cell and is able to penetrate into the target cell. If a targetintracellular enzyme is present in the target cell, the enzyme is ableto remove the caging component of the caged substrate, thus producing anun-caged substrate. The un-caged substrate can then react with thereporter molecule inside of the cell, and the product of this reactionresults in a detectable signal. Herein, target cells can includeeukaryotic and prokaryotic cell targets and associated enzymes,including, for example, β-lactamases in S. aureus. The delivery of thereporter molecule-expressing nucleic acid containing the recombinant DNAcan by performed by a biologic or biologic systems including but notlimited to liposomes, virus-like particles, transduction particlesderived from phage or viruses, and conjugation. Further, variousreporter molecules and caged substrates can be employed as thosedescribed in Daniel Sobek, J. R., Enzyme detection system with cagedsubstrates, 2007, Zymera, Inc. In certain aspects, a reportermolecule-expressing vector can be carried by a NRTP, such that thevector can be delivered into a bacterial cell. The reporter molecule tobe expressed can be Renilla luciferase, and the caged substrate can beRenilla luciferin that is caged, such that a β-lactamase enzyme that isendogenous to the target cell is able to cleave the caging compound fromthe caged luciferin and release un-caged luciferin. In this manner, whena target cell that contains the β-lactamase is exposed to the NRTP andcaged luciferin, the cell will exhibit a luminescent signal that isindicative of the presence of the β-lactamase present in the cell.

If the target enzyme is present in a cell expressing bacterialluciferase and the caged fatty aldehyde is applied to the samplecontaining the cell, the enzyme can un-cage the fatty aldehyde, allowingfor a luminescence reaction to occur, which produces a detectableluminescence signal. If the target enzyme is not present in the cell,the system does not produce a detectable signal.

Here, a reporter system is developed for detecting the presence ofcarbapenemase in cells. Decanal is the caged molecule, caged by ameropenem-based molecule, resulting in a carbapenem-caged decanal.Approximately 100-200 mg of the decyl acetal of meropenem (i.e., thecarbapenem-caged decanal) were synthesized by the scheme shown in FIG.5. An alternative mechanism of synthesis of the decyl acetal ofmeropenem without the 1-beta-methyl group is performed as shown in FIG.6.

To detect the presence of the carbapenemase, an Enterobacteriaceae-basedNRTP including a luciferase reporter system is produced (as described inPCT/US2014/026536, Example 1). The NRTP and carbapenem-caged decanal areadded to a sample that is suspected of containing Enterobacteriaceaethat expresses a carbapenemase. If Enterobacteriaceae is present in thesample, the reporter system causes the expression of bacterialluciferase in the cell. If the cell also expresses a carbapenemase, thenthe carbapenemase un-cages the decanal molecule and allows it to mediatea luminescence reaction via the expressed bacterial luciferase, causingthe production of a detectable luminescent signal. If the cell does notcontain the carbapenemase, the system does not produce a detectablesignal. In this manner, the system is able to report on the presence ofa carbapenemase in the Enterobacteriaceae cell. The reporter assay isalso run combined with a carbapenem to determine whether theEnterobacteriaceae is susceptible or non-susceptible to the carbapenem.This system is further used to determine the mechanism involved incarbapenem susceptibility. In the assay, the reporter assay is run todetermine if Enterobacteriaceae is present. The reporter assay is alsorun combined with a carbapenem to determine if the organism isnon-susceptible to the carbapenem. The reporter assay is also run usinga carbapenem-caged substrate to determine if the organism expresses acarbapenemase.

If the above reporter assay determines that the target organism ispresent and is non-susceptible to the carbapenem, the reporter assay isperformed again combined with various reagents: the assay is run withthe carbapenem and a β-lactamase inhibitor such as boronic acid todetermine if the non-susceptible organism becomes susceptible in thepresence of the β-lactamase inhibitor. The reporter assay is also runwith the carbapenem and a chelator such as EDTA to determine if thenon-susceptible organism becomes susceptible in the absence of metal.

Table 2 depicts results produced by the assay, where ‘x’ denotes apositive result by the reporter assay. Sample 1 does not contain thetarget organism. Sample 2 contains the target organism that issusceptible to the carbapenem and it does not express a carbapenemase.Sample 3 contains the target organism, is non-susceptible to thecarbapenem, the β-lactamase inhibitor inhibits the non-susceptibleresult, the chelator does not inhibit the non-susceptible result, andthe organism expresses a carbapenemase; based on these results, theorganism likely expresses a Class A carbapenemase. Sample 4 contains thetarget organism, is non-susceptible to the carbapenem, the β-lactamaseinhibitor does not inhibit the non-susceptible result, the chelator doesinhibit the non-susceptible result, and the organism expresses acarbapenemase; based on these results, the organism likely expresses aClass B carbapenemase. Sample 5 contains the target organism, isnon-susceptible to the carbapenem, the β-lactamase inhibitor does notinhibit the non-susceptible result, the chelator does not inhibit thenon-susceptible result, and the organism expresses a carbapenemase;based on these results, the organism may express a Class D carbapenemaseor multiple carbapenemases including Class D and Class A or Class B, orClass A and Class B. Sample 6 contains the target organism, isnon-susceptible to the carbapenem, the β-lactamase inhibitor does notinhibit the non-susceptible result, the chelator does not inhibit thenon-susceptible result, and the organism does not express thecarbapenemase; based on these results, the non-susceptible phenotype ofthe organism may be due to a mechanism such as AmpC over-expression andporin mutations. Sample 7 contains the target organism, is susceptibleto the carbapenem and expresses a carbapenemase; based on these results,the organism is a carbapenem-susceptible, carbapenemase-expressingorganism. Using the assay described above, the carbapenem susceptibilityphenotype of the Enterobacteriaceae in several samples 1-7 wasdetermined.

TABLE 2 Results of carbapenem susceptibility mechanism determinationassay Reporter + Reporter + Reporter + Antibiotic + Antibiotic +Reporter + Reporter Antibiotic Inhibitor EDTA Reagent Result Sample 1 NANA NA NA No target cell present Sample 2 x NA NA Susceptible Sample 3 xx x x Non-susceptible, expresses Class A carbapenemase Sample 4 x x x xNon-susceptible, expresses Class B carbapenemase Sample 5 x x x x xNon-susceptible, expresses Class D carbapenemase or Class A & Bcarbapenemases Sample 6 x x x x Non-susceptible, does not express acarbapenemase Sample 7 x NA NA x Susceptible and expresses carbapenemase

Further testing includes the addition of cloxacillin as an inhibitor ofAmpC-mediated non-susceptibility. In this case, the addition oncloxacillin can allow for confirmation that a carbapenemase is notresponsible for the non-susceptibility result of Sample 6 in which case,the addition of cloxacillin would cause Sample 6 to exhibit anon-susceptible result with the addition of the carbapenem.

Example 4: A Novel Systems-Based Molecular Diagnostic for CarbapenemSusceptibility Testing and Resistance Mechanism Determination

Background: Carbapenem resistance in Gram-negative bacteria has become aserious clinical issue with limited diagnostic and treatment options.Novel antimicrobials such as the recently approved Avycaz (Actavis), andthe in-development Relebactam (Merck) and Carbavance (The MedicinesCompany) have shown activity against Class A carbapenemases (e.g. KPC)though not against Class B carbapenemases (e.g. NDM-1). Thus, theability to quickly detect viable Gram-negative bacteria, determine theirsusceptibility to carbapenems, and distinguish between carbapenemresistance mechanisms could aid physicians in considering suchtherapeutic options in the future. We have developed Smarticles™technology, a live cell luminescence assay for detection andsusceptibility testing directly from clinical samples, as describedherein, e.g., at least at Examples 1-4, and throughout the detaileddescription. In this study, a Smarticles™ assay was evaluated for itsability to detect carbapenem resistant E. coli and distinguish betweenNDM-1 and KPC-expressing organisms.

Methods: 26 clinical isolates of E. coli, Klebsiella spp., Enterobacterspp., Citrobacter spp., Proteus spp., and S. marcescens were evaluatedusing the Smarticles™ assay and ertapenem disk diffusion. CRE isolatesincluded organisms expressing blaKPC and blaNDM-1. The Smarticles™ assaywas used with the addition of the chelating agent edetic acid (EDTA) andβ-lactamase inhibitor boronic acid to scrutinize clinical isolates of E.coli expressing NDM-1 and KPC.

Results: The Smarticles™ assay demonstrated the ability to distinguishbetween ertapenem susceptible and non-susceptible Enterobacteriaceae in2 hours. The Smarticles™ assay was also able to distinguish betweenNDM-1 and KPC-expressing E. coli where, in the presence of EDTA, theassay signal was inhibited in NDM-1-expressing E. coli and not inhibitedin KPC-expressing E. coli and, in the presence of boronic acid, theassay signal was inhibited in KPC-expressing E. coli and not inhibitedin NDM-1-expressing E. coli.

CONCLUSION

The ability of the Smarticles™ assay to detect carbapenem-resistantbacteria and distinguish among carbapenem-resistance mechanisms has thepotential to aid in treatment guidance for novel antibiotics that havebeen shown to have activity against a targeted group of resistancemechanisms.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

What is claimed:
 1. A kit for determining a mechanism of antimicrobialresistance for a microorganism to an antimicrobial agent comprising: anantimicrobial agent, wherein the antimicrobial agent is a compound thatkills, inhibits the growth of, or otherwise compromises the viability ofthe growth of one or more microorganisms; a non-replicative transductionparticle (NRTP) comprising a reporter nucleic acid molecule encoding areporter molecule; a caged substrate having a structure of

and capable of entering the microorganism and becoming an un-cagedsubstrate in the presence of an enzyme related to a mechanism ofresistance to the antimicrobial agent, wherein the un-caged substratereacts with the reporter molecule to produce a detectable signal,wherein detection of the detectable signal confirms a presence of themicroorganism in the sample; and instructions for using theantimicrobial agent, the NRTP, and the caged substrate to determine themechanism of antimicrobial resistance for the microorganism to theantimicrobial agent based on the presence or absence of a detectableindication of viability associated with the microorganism when themicroorganism is in contact with the antimicrobial agent, the NRTP andthe caged substrate, wherein the presence of a detectable indication ofviability indicates that the microorganism is viable and that the enzymerelated to the mechanism of resistance to the antimicrobial agent isexpressed by the microorganism, and wherein the absence of an indicationof viability indicates that the microorganism is not viable and that theenzyme related to the mechanism of resistance to the antimicrobial agentis not expressed by the microorganism.
 2. The kit of claim 1, whereinsaid reporter molecule is expressed from a reporter gene wherein saidreporter gene is selected from the group consisting of genes encodingenzymes mediating luminescence reactions and genes encoding enzymesmediating colorimetric reactions.
 3. The kit of claim 2, wherein saidgenes encoding enzymes mediating luminescence reactions comprise genesencoding luciferases.
 4. The kit of claim 1, wherein said microorganismis a prokaryote or a eukaryote.
 5. The kit of claim 4, wherein saidprokaryote is a Gram-negative bacteria or a Gram-positive bacteria. 6.The kit of claim 1, wherein said microorganism is any one ofStaphylococcus spp., Enterobacteriaceae, Enterococcus spp.,Streptococcus spp., Acinetobacter spp., Pseudomonas spp.,Stenotrophomonas spp., Mycobacterium spp., or Candida.
 7. The kit ofclaim 1, wherein said mechanism of antimicrobial resistance comprisescarbapenemase activity.
 8. The kit of claim 7, wherein saidcarbapenemase activity is from a Class A carbapenemase, a Class Bcarbapenemase, a class D carbapenemase, or a combination thereof.
 9. Thekit of claim 1, wherein said antimicrobial resistance comprisesresistance to a carbapenem.
 10. The kit of claim 1, wherein said enzymeis a carbapenemase.